This project is concerned with the relation of the structure to the function of dihydrofolate reductase (dihydrofolate plus NADPH yields tetrahydrofolate plus NADP ion) from Methotrexate (MTX)-resistant bacterial, chicken liver, and MTX-resistant sublines of human lymphoblastoid cells. Using several techniques of protein chemistry, the problems to be investigated include: (a) equilibrium binding studies of substrates, coenzymes and inhibitors to enzyme using circular dichroic (CD), uv difference, fluorometric and equilibrium dialysis techniques, (b) chemical modification of specific amino-acid residues on the protein and measurement of the effect of thse modifications upon the binding of substrates and inhibitors and upon enzymic activity; (c) amino-acid sequence determination of enzymes from two MTX-resistant sublines of human lymphoblastoid cells; (d) examination of inhibition properties of selected quinazoline derivatives and other compounds towards bacterial, chicken liver and human enzymes; (e) determination of transport characteristics of selected quinazoline drugs in human lymphoblastoid cell lines; (f) design and evaluation of active site-directed irreversible inhibitors of dihydrofolate reductase.